Galectin‐9 alleviates acute graft‐versus‐host disease after haplo‐hematopoietic stem cell transplantation by regulating regulatory T cell/effector T cell imbalance

Abstract Background Acute graft‐versus‐host disease (aGVHD) arises from the imbalance of host T cells. Galectin‐9 negatively regulates CD4 effector T cell (Th1 and Th17) function by binding to Tim‐3. However, the relationship between Galectin‐9/Tim‐3 and CD4+ T subsets in patients with aGVHD after Haplo‐HSCT (haploidentical peripheral blood hematopoietic stem cell transplantation) has not been fully elucidated. Here, we investigated the role of Galectin‐9 and CD4+T subsets in aGVHD after haplo‐HSCT. Methods Forty‐two patients underwent Haplo‐HSCT (26 without aGVHD and 16 with aGVHD), and 20 healthy controls were included. The concentrations of Galectin‐9, interferon‐gamma (IFN‐γ), interleukin (IL)‐4, transforming growth factor (TGF)‐β, and IL‐17 in the serum and culture supernatant were measured using enzyme‐linked immunosorbent assay or cytometric bead array. The expression levels of Galectin‐9, PI3K, p‐PI3K, and p‐mTOR protein were detected by western blot analysis. Flow cytometry was used to analyze the proportions of CD4+ T cell subsets. Bioinformatics analysis was performed. Results In patients with aGVHD, regulatory T (Treg) cells and Galectin‐9 decreased, and the Th1, Th17, and Treg cells were significantly imbalanced. Moreover, Treg and Galectin‐9 were rapidly reconstituted in the early stage of patients without aGVHD after Haplo‐HSCT, but Th17 cells were reconstituted slowly. Furthermore, Tim‐3 upregulation on Th17 and Th1 cells was associated with excessive activation of the PI3K/AKT pathway in patients with aGVHD. Specifically, in vitro treatment with Galectin‐9 reduced IFN‐γ and IL‐17 production while augmenting TGF‐β secretion. Bioinformatics analysis suggested the potential involvement of the PI3K/AKT/mTOR pathway in aGVHD. Mechanistically, exogenous Galectin‐9 was found to mitigate aGVHD by restoring the Treg/Teffs (effector T cells) balance and suppressing PI3K. Conclusion Galectin‐9 may ameliorate aGVHD after haplo‐HSCT by modulating Treg/Teffs balance and regulating the PI3K/AKT/mTOR pathway. Targeting Galectin‐9 may hold potential value for the treatment of aGVHD.


| INTRODUCTION
2][3] The incidence of aGVHD is higher in patients with stem cell transplants from HLA-mismatched and unrelated donors. 2,3GVHD is characterized by the excessive activation of effector T cells (Teffs), including Th1, Th17, and cytotoxic CD8 + T cells, which can result in the excessive secretion of cytokines, and ultimately induce inflammatory injury to target organs. 4,5alectin-9 is a widely expressed soluble molecule that can bind to Tim-3 and negatively regulate the immunity function of Teffs. 6By binding to Tim-3 on Th1 and Th17 cells, it induces apoptosis and/or inhibits cell differentiation. 7The Tim-3/Galectin-9 pathway acts as a negative regulator of effector CD4 + and CD8 + T cells, thereby prolonging the lifespan of allogeneic skin grafts. 6,8,9Our previous study found that in patients with aGVHD who had received haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-HSCT), Tim-3 expression was upregulated while serum Galectin-9 was downregulated. 10 However, the underlying mechanism of Tim-3 and Galection-9 in aGVHD remains unclear.
Shayan et al. found that the PI3K/AKT/mTOR pathway mediated cross-talk between Tim-3 and PD-1. 113][14][15] The PI3K/AKT/mTOR pathway plays a crucial role in T cell activation and function.This pathway regulates many cellular events of T cells, including proliferation, survival, migration, and metabolism. 166][27] Herrero-Sánchez et al. 27 demonstrated that targeting the PI3K/AKT/mTOR pathway inhibited T cell activation and thus prevented GVHD development.Studies have shown that the rapid reconstitution of regulatory T (Treg) cells and the upregulation of TGF-β and IL-10 after haplo-haplo-HSCT can induce immune tolerance and decrease the risk of aGVHD. 28,29The imbalance between Treg and Teffs is a major contributor to aGVHD. 30,31The key to alleviating aGVHD is to restore the Treg/Teffs balance.To our knowledge, the relationship between PI3K/AKT/ mTOR and Treg/Teffs balance in patients with aGVHD after haplo-HSCT has not been reported.Therefore, we evaluated whether Galectin-9 could modulate Treg/Teffs balance to ameliorate aGVHD after haplo-HSCT.The PI3K/AKT/mTOR pathway was also analyzed and discussed.The findings suggest that targeting Galectin-9 could potentially facilitate the treatment of aGVHD.

| Study participants
This study included 42 patients who underwent Haplo-HSCT for hematological malignancies from December 2018 to August 2022.All transplantations were successful, with donor cell chimerism rates exceeding 95%.Hematopoietic reconstitution was achieved in all patients within 20 days posttransplantation, and blood samples were collected on Days 30, 45, 60, and 90 following transplantation.Peripheral blood was collected immediately upon occurrence of aGVHD before Day 30 posttransplantation.Meanwhile, healthy controls (HC) (n = 20) were also enrolled, and their peripheral blood samples were also collected.We excluded patients who died or relapsed within 3 months after transplantation.Written informed consent was provided.This study was approved by the Ethical Committee of Xinjiang Medical University (20120220-126).

| Conditioning regimen and mobilization, collection, and transfusion of peripheral blood hematopoietic stem cells (PBSCs) from donors
The precondition and mobilization regimens were performed according to our previous studies. 28,29The classic Ara-c+Bu/Cy+ATG conditioning regimen and the Bu/Cy +ATG regimen were administered.Granulocyte colonystimulating factor (7-10 µg/kg.d)was used to mobilize peripheral PBSCs from donors, which were then collected on the 5th and 6th day post mobilization and infused to the recipients on the same day of collection.The infusion volume of mononuclear cells was (13.44 ± 5.23) × 10 8 /kg, and that of CD34+ cells was (7.53 ± 2.78) × 10 6 /kg.

| aGVHD prophylaxis
An enhanced GVHD prophylaxis regimen was used, including CsA, Tac, MTX, anti-CD25 mAb, MMF, DXMS, and MP, as previously described. 28,29The aGVHD diagnosis and grading followed the Seattle criteria 32 and the Chinese expert consensus on allogeneic hematopoietic stem cell transplantation for the treatment of hematological diseases (Ⅲ)-acute versus host disease. 33fter confirmation of aGVHD, methylprednisolone (1 mg/kg) was used as the first line of treatment, with the dose adjusted based on the severity of aGVHD, and other immunosuppressants were used as necessary.

| Flow cytometry
The proportions of CD4 + T subsets were analyzed with flow cytometry.CD4 + T subset biomarkers were selected as previously described. 34For the analysis of Th17, Th1, and Th2 cells, peripheral blood samples (100 μL) were incubated with CD4-FITC, CCR6-PerCP-Cy TM 5.5, CXCR3-APC, and Tim-3-PE fluorescent-labeled antibodies.For the analysis of Treg cells, the peripheral blood samples (100 μL) were incubated with CD4-FITC, CD25-APC, CD127-PerCP-Cy TM 5.5, and Tim-3-PE fluorescent-labeled antibodies.IgG-PE was used as the isotype control.BD Biosciences provided the antibodies and reagents.The antibody incubation was performed at 4°C for 20 min in the dark, followed by red blood cell lysis for 5 min in the dark.After centrifugation and washing, the cells were resuspended and detected on a flow cytometer (Canto II, BD Biosciences).Fluorescence signals were excited at 488 nm and acquired using the 530/30-A channel for FITC, 695/40-A channel for PerCP-Cy™5.5, and 575/25-A channel for PE.For APC, fluorescence signals were excited at 633 nm and acquired using the 670/30-A channel.At least 50,000 cells per sample were analyzed.Data analysis was conducted with the Flow Jo software (Tree Star) and Kaluza software (Beckman Coulter, Inc.).Lymphocytes were first gated using SSC and FSC.Then, CD4 + T cells were gated, followed by the analysis of each cell subset, including Th1 cells (CXCR3 + CCR6 − CD4 + ), Th2 cells (CXCR3 − CCR6 − CD4 + ), Th17 cells (CXCR3 − CCR6 + CD4 + ), and Treg cells (CD25 hi CD127 low CD4 + ).

| Tim-3 + CD4 + T cell sorting
PBMCs were isolated from six patients with aGVHD using a lymphocyte separation solution (TBD Science).Subsequently, the Negative Selection CD4 + T-cell sorting kit (STEMCELL Technologies) was used to enrich CD4 + T cells.The sorted CD4 + T cells were re-suspended to 5 × 10 7 /mL and incubated with Tim-3-PE antibody (BD, Clone:7D3, 2 µg/mL) in the dark for 15 min.Then, the incubation with 100 μL of EasySep® PE Selection Cocktail (STEMCELL Technologies) and 50 μL of EasySep® Magnetic Nanoparticles (STEMCELL Technologies) was performed.Finally, Tim-3 + CD4 + T cells were evaluated by flow cytometry, and their purity was over 90%.

| CBA
The serum was isolated from the peripheral blood samples.Interleukin (IL)-17, interferon-gamma (IFN-γ), and IL-4 levels in the serum and cell culture supernatant were determined using the human Th1/Th2/Th17 CBA Kits (BD).Finally, the samples were detected on a flow cytometer (Canto II, BD Biosciences).The data were analyzed using FCAP Array software version 3.0 (BD).

| Enzyme-linked immunosorbent assay (ELISA)
The levels of transforming growth factor (TGF-β) and Galectin-9 in the serum and culture supernatant were determined using ELISA kits (Thermo Fisher) (TGF-β1: Cat# BMS249-4; Galectin-9: Cat# EH206RB), following the kit instructions.Blank control wells were set up.Finally, the absorbance of each well at 450 nm was measured on a microplate reader (Multiskan Go; Thermo Fisher).The levels of TGF-β and Galectin-9 were calculated according to the standard curves.

| Identification of differentially expressed genes
Peripheral blood samples were collected from patients with and without aGVHD (n = 3 each).PBMCs were isolated.The RNA was isolated from PBMCs and subjected to Illumina sequencing (Novogene).The sequencing results were analyzed using the DESeq. 2 software (1.20.0), and differentially expressed genes were screened using |log 2 fold change [FC] | > 2 and p < .05.

| Statistical analysis
To describe the measurement data, we calculated the mean and standard deviation.We employed the Student's t-test or analysis of variance followed by the LSD method, for the comparison of the data.Data with a heterogeneity of variance were analyzed with the rank sum test.The Spearman correlation test was used for correlation analysis.The data were processed with SPSS (version 22, SPSS Statistics/IBM Corp).p < .05indicates a significant difference.1A,B.Our previous study found that reconstitution of CD4 + T cells began within 30 days after transplantation and gradually increased from 60 to 90 days posttransplantation. 28 However, the CD4 + T cell counts remained lower than those of healthy individuals.In this study, we found reconstitution of Treg and Th1 cells began 30 days after transplantation, and there was no significant difference at 60-90 days between each group and HC group (all p > .05, Figure 1C).Reconstitution of Th17 cells began 45 days after transplantation, and Th17 cells gradually increased and returned to normal levels 60 days after transplantation.

| Genes associated with PI3K/AKT/ mTOR signal pathway are upregulated after Haplo-HSCT
The aGVHD is associated with T-cell activation and signaling pathway activation. 26,27Here, we compared the transcriptomic profiles of patients with and without aGVHD (n = 3 each).The volcano plot showed 435 upregulated and 455 downregulated transcripts in patients with aGVHD (Figure 4A).GO analysis found that the genes with differential expression were mainly enriched in 115 GO terms of biological process (such as extracellular matrix organization) (p < .05), in 9 GO terms of cellular component (such as azurophil granule) (p < .05),and in 19 GO terms of molecular function (such as cytokine receptor activity) (p < .05)(Figure 4B).KEGG analysis showed that 21 pathways were significantly upregulated in aGVHD patients, including cytokinecytokine receptor interaction, IL-17 signaling pathway, mTOR signaling pathway, PI3K-AKT signaling pathway, and so forth (Figure 4C).Among them, the role of the PI3K/AKT/mTOR signaling pathway in aGVHD was significant (Figure 4D).Thus, it is necessary to further study the relationship between PI3K/AKT/mTOR and aGVHD.aGVHD, acute graft-versus-host disease; CBA, cytometric bead array; ELISA, enzyme-linked immunosorbent assay; GO, Gene Ontology; IFN-γ, interferon-gamma; IL, interleukin; KEGG, Kyoto Encyclopedia of Genes and Genomes; PBS, phosphate-buffered saline; TGF-transforming growth factor.

| DISCUSSION
Both "cytokine storm" and excessive activation of T cells are considered the main contributors to aGVHD. 35,36revious studies have found that Th1 cytokine IFN-γ in aGVHD patients is significantly upregulated and then decreased obviously after treatment, suggesting that serum IFN-γ level is likely associated with aGVHD. 28,29im-3 on activated T cells could bind to Galectin-9 and inhibit Teffs. 37,38In this study, we found that there was an early and rapid immune reconstitution of Treg cells and Galectin-9 levels in patients without aGVHD after Haplo-HSCT, which may effectively prevent aGVHD.When aGVHD is present, the Galectin-9/Tim-3 negative pathway is not yet formed due to Galectin-9 deficiency.Furthermore, in patients with aGVHD, there was high expression of Tim-3 on effector CD4 + T cells, and the production of cytokines (such as IL-17 and IFN-γ) was elevated after the activation of the PI3K/AKT/mTOR pathway.Finally, exogenous Galectin-9 inhibited the activation of PI3K/AKT, thus alleviating aGVHD by reversing Treg/Teffs imbalance in vitro.
Restoration of innate and adaptive immunity is necessary for effective graft versus leukemia and for preventing post-transplantation infections. 39,40Ito et al. found that low-dose Thymoglobulin suppressed the restoration of naïve T cells after allo-HCT, but not the immune reconstitution of T cells. 41Here, our data demonstrated that hematopoietic reconstitution was achieved in all patients who received Haplo-HSCT.Although ATG was used, Treg and Th1 cells in patients without aGVHD had a faster immune reconstitution in the early post-transplantation period.Reconstruction of Treg can effectively prevent aGVHD.Recently, it has been revealed that early CD4 + T cell immune reconstitution is related to patient survival with moderate to severe aGVHD. 42Cytokines secreted by different subsets of T cells after HSCT are involved in aGVHD. 43n this study, we demonstrated that patients who underwent Halpo-PBSCT and were diagnosed with aGVHD exhibited an increase in Th1 and Th17 cells, but a reduction in Treg cells.Moreover, these patients had a significant decrease in the levels of Galectin-9 and TGF-β.This suggests that among aGVHD patients who underwent Halpo-HSCT, there is an imbalance not only between Th1 and Th2 cells but also between Treg and Th17 cells, as well as an abnormal pattern of cytokine ratios IFN-γ/IL-4 and TGF-β/IL-17.In a mouse model of aGVHD, the overexpression of Tim-3 was observed in CD4 + T and CD8 + T cells from the spleen and liver. 44ansen et al found that Tim-3 level was related to aGVHD severity. 45Here, our results demonstrated that there was Tim-3 overexpression on Th1 and Th17 cells in patients with aGVHD after Haplo-HSCT, suggesting that Th1 and Th17 cells may be abnormally activated in patients with aGVHD.Tim-3 binding to Galectin-9 can lead to intracellular calcium efflux and cell aggregation, which eventually results in the programmed cell death of Th17 and Th1 cells. 7,37The interaction of Galectin-9 and Tim-3 can negatively regulate the response of Th1 and Th17 cells, and induce peripheral tolerance. 7,46Nevertheless, in this study, Galectin-9 was reduced significantly in patients who had aGVHD.We hypothesize that Galectin-9 deficiency may lead to the absence of the Tim-3/Galectin-9 negative signaling pathway, thereby affecting Tim-3's ability to exert a negative immunoregulatory function.
Studies have shown that Galectin-9 can also interact with PD-1 and VISTA. 47,48Galectin-9 facilitates T-cell apoptosis through the cross-linking of Tim-3, while the co-expression of PD-1 attenuates Galectin-9/Tim-3induced apoptosis by promoting the formation of Tim-3/Galectin-9/PD-1 lattices in cancer. 47Additionally, secreted Galectin-9 alone can enhance PI3K activity in T cells, and downregulation results from the synergistic effect of Galectin-9 with other immune checkpoint proteins such as VISTA (V-domain immunoglobulin suppressor of T cell activation) and PD-L1, 49,50 or small molecular weight compounds like L-Kynurenine. 51PD-1 can activate the protein phosphatase SHP-2, which in turn downregulates the phosphorylation of crucial upstream kinases in T cells, resulting in decreased PI3K activity and thus regulating the PI3K/Akt/mTOR pathway. 52In this study, we found that 455 genes were downregulated and 435 genes were upregulated in patients with aGVHD.Additionally, the PI3K/AKT signal pathway was upregulated in patients with aGVHD.Through in vitro experiments, we revealed for the first time that in aGVHD patients, exogenous Galectin-9 could bind to Tim-3 on the cell surface and downregulate the phosphorylation level of PI3K in CD4 + T cells.This may affect the PI3K/Akt/mTOR pathway, thus inhibiting the function of effector CD4 + T cells.However, whether Galectin-9, after haplo-HSCT binds synergistically with PD-1 and VISTA to downregulate the phosphorylation of upstream key kinases in T cells and inhibit PI3K activity is not yet clear, and further in-depth research is warranted.
The Tim-3/Galectin-9 pathway plays a crucial role in promoting immunological tolerance in organ transplantation. 53In a study on mouse skin transplantation, exogenous Galectin-9 protein downregulated the Tim-3 + Th1 immune response, alleviated inflammation, and ultimately prolonged the survival time of skin grafts. 8alectin-9 can inhibit T cell proliferation and specifically reduce IFN-γ secretion in vitro. 7Another study found that Galectin-9 (+) Th cells affected Th17/Treg cell balance by secreting Galectin-9, similar to exogenous Galectin-9. 54In our study, we found that exogenous rhGalectin-9 significantly reduced the phosphorylation levels of PI3K and cytokine levels of IL-17 and IFN-γ.High doses of Galectin-9 can inhibit Teffs, and thus a low serum level of Galectin-9 poses a potential risk factor for aGVHD. 55In this study, activation of Tim-3 with rhGalectin-9 led to an enhanced function of Treg cells and caused a significant elevation of TGF-β in vitro, which may reverse the Treg/Teffs imbalance.
Our study has some limitations.For example, the study had a small sample size.Moreover, the interaction of Galectin-9 with PD-1 and its synergistic effects with Tim-3 was not studied through in vitro experiments.Further studies are warranted.tolerance utilizing the Galectin-9/Tim-3 pathway may become a novel immune approach for the treatment and prevention of aGVHD in the future.

3 . 3 | 3 . 4 |
On 30-90 days post-transplantation, the number of Th2 cells was lower in comparison to the HC group (p < .05),indicating delayed immune reconstitution of Th2 cells.Hence, Treg and Th1 cells have faster immune reconstitution in the early posttransplantation period, Th17 cells begin to increase at 45 days after transplantation, and Th2 cells are relatively lagging in immune reconstitution.The change of cytokines after transplantation in patients without aGVHD CD4 + T cell subsets exert biological effects by secreting different cytokines.In this study, the cytokines in the serum of patients without aGVHD were measured using CBA and ELISA.As shown in Figure1D, compared with HC, the IL-4, IFN-γ, Galectin-9, IL-17, and TGF-β in the serum of patients without aGVHD were markedly reduced on day 30 post-transplantation. A gradually increasing trend in serum TGF-β was observed on days 45 and 60 post-transplantation.The level of Galectin-9 in the serum increased on day 45, with no statistically significant difference compared to the control group (p > .05).Furthermore, the serum IL-17, IFN-γ, and IL-4 were significantly lower than those in HC on days 30 and 45 post-transplantation (p < .05).However, no significant differences between patients and HC were observed at other time points (p > .05).Therefore, the alterations of serum cytokines are in line with those of Th1, Th2, Treg, and Th17 cells.The serum level of Galectin-9 has a slightly faster recovery than IL-4, TGF-β, IFN-γ, and IL-17.Decreased Treg and imbalanced Treg/Th17 in peripheral blood of patients with aGVHD Changes in CD4 + T cell subsets were analyzed using flow cytometry in 26 patients without aGVHD (30 days posttransplantation) and 16 patients with aGVHD (31 days posttransplantation).The results revealed that the Th1 and Th17 cell proportions were significantly higher in aGVHD patients (aGVHD(+)) than in patients without aGVHD (aGVHD(−)) (p < .01)(Figure2A,B).Conversely, the aGVHD(−) patients had significantly increased Treg cell proportion than the aGVHD(+) patients (p < .01).The aGVHD(+) and aGVHD(−) patients (p > .05)did not differ significantly in Th2 cell proportion.The aGVHD(+) patients exhibited significantly elevated Th17/Treg and Th1/Th2 ratios than aGVHD(−) patients and HC (Figure2D).The analysis of cytokine levels revealed statistically elevated levels of IFN-γ and IL-17 in aGVHD(+) patients compared to aGVHD(−) patients (p < .01).Compared with T A B L E 1 Clinical characteristics of patients with related HLA haploidentical allogeneic peripheral blood hematopoietic stem cell transplantation (haplo-HSCT).

Following
Haplo-HSCT, the early immune reconstitution of Treg cells and the increased secretion of Galectin-9 and TGF-β can prevent the occurrence of aGVHD.Importantly, we have demonstrated that Galectin-9 can inhibit the activation of CD4 + T cells in two ways.First, Galectin-9 may inhibit the activation of the PI3K/AKT pathway and downregulate the expression of PI3K in Tim-3 + CD4 + T cells of aGVHD patients.Secondly, TGF-β promotes the differentiation of Treg cells through autocrine secretion.On the other hand, TGF-β induces the expression of Galectin-9 in a paracrine manner, reversing the imbalance of Treg/Teff and thereby inducing immune tolerance and preventing the occurrence of aGVHD (Figure 5).Reconstitution of immune F I G U R E 5 Schematic diagram illustrating the hypothesis of signaling regulation in aGVHD.In patients with aGVHD, the expression of Tim-3 is significantly increased.Galectin-9 binding to Tim-3 may inhibit the activation of the PI3K/AKT pathway and enhance the function of Treg cells.On the other hand, TGF-β promotes the differentiation of Treg cells through autocrine secretion, while TGF-β induces the expression of Galectin-9 in a paracrine manner.The increased Treg cells can inhibit the activation of Th1 and Th17 cells by secreting TGF-β, thus alleviating aGVHD by inducing immune tolerance.aGVHD, acute graft-versus-host disease; IFN-γ, interferon-gamma; IL, interleukin; TGF-transforming growth factor.

Table 1
Flow cytometry assessed the reconstitution of peripheral CD4 + T cell subsets at 30, 45, 60, and 90 days after Haplo-HSCT in patients without aGVHD.The gating strategy is shown in Figure